The invention pertains to the field of characterization and analysis of any material between 2 and 10,000 kilodaltons in molecular weight especially biopolymers or other polymers using classical light scattering. More particularly, the invention pertains to an improvement over classical low angle light scattering detectors and multiangle classical light scattering detectors designed to determine molecular weight of particles emerging from a separation device such as a liquid chromatography column.
The molecular weight of a particle eluting from a liquid chromatography column or other separation process is a very useful thing to know for both characterization of the particle, analysis of the chromatogram and for process monitoring and control. For example, suppose a biological process generates a sample having several different biological proteins of interest and scientists want to separate and identify them by molecular weight for further experiments regarding their utility in treatment of disease or for diagnostic purposes. There are no liquid chromatography columns that separate proteins by molecular weight, but there are columns that separate particles by size. However, size is poor indicator of molecular weight because a biological protein in the natural globular form and the same protein in the denatured state have vastly different sizes but the same molecular weight. It would be useful for separation of the various proteins as they elute from a liquid chromatography column based upon, for example, size, to know the molecular weight of the proteins that cause each peak in the chromatograph. The molecular weight signal can then be used to control a collection system into which the protein stream flows such that the different proteins are captured in different containers.
Some detectors exist in the prior art which detect the difference in refractive index of the output stream of a liquid chromatography column. However, refractive index is not a good indicator of molecular weight because.
Another application where knowledge of the molecular weight of the particles eluting from a liquid chromatography (hereafter LC) column is analysis of chromatograms. In some situations, peaks in a chromatogram generated using a ultraviolet detector (hereafter UV) cannot be deciphered in terms of what types of particles caused the peaks. If a classical light scattering detector is used to generate another chromatogram, the two chromatograms may be compared and the differences between the mass of particles which caused the different peaks in the UV chromatogram may be easily determined.
In the prior art, molecular weight determinations have been difficult and based upon instruments that were designed in 1970 based upon a view of the mathematics of classical light scattering which is not optimized for small particles having sizes less than .lambda./4 where .lambda. is the incident light wavelength such as biopolymers. Specifically, there is a relationship which mathematically relates molecular weight, Rayleigh scattering, weight concentration of the particles causing the scattering, a size factor called P and another physical characteristic called the second viral coefficient which pertains to the volume excluded by a particular biological protein based upon its characteristics. This second viral coefficient, A, if large, means that a particular biological protein excludes other proteins from a very large volume around it. If A is negative, it means that this biological protein tends to attract other proteins and form agglomerations. More specifically, the relationship is: ##EQU1## where K=an optical constant relating wavelength, refractive index of the solution and change in refraction index of the solution over time among other things and which can be measured empirically for any given system,
R=the "specific Rayleigh constant" or the "specific Rayleigh ratio", PA1 M.sub.w =the weight averaged molecular mass of the scatterers, PA1 P(.theta.)=a size parameter which corrects equation (1) for the effects of multiple intraparticle scattering, PA1 A.sub.2 and A.sub.3 =the second and third viral coefficients, respectively, and PA1 C=the weight concentration of the scattering particles (hereafter the scatterers). PA1 n=the index of refraction PA1 R.sub.9 =the radius of gyration of the scatterer; PA1 .theta.=the scattering angle, i.e., the angle between the incident light and the scattered light, PA1 .lambda.=the wavelength of the incident light. PA1 I.sub.s /I.sub.o =the intensity of the high angle scattered light at some angle between 35.degree. and 145.degree. relative to the intensity I.sub.o of the light incident on the flow cell (this ratio cancels noise caused by variations in the output intensity of the light source), PA1 C=the weight concentration, PA1 M.sub.w =the weight averaged molecular weight of the scatterers in the portion of the output stream for which the weight concentration C was calculated, and PA1 B=an optical constant which is different for each system and which is measured either empirically using several different types of particles of known weight averaged molecular weight of absolutely using a solution of known scattering ability such as Toluene.
One of the difficulties faced by workers in the art lies in the P factor in equation (1). Specifically, P is given by: ##EQU2## where P(.theta.).sup.-1 =the size factor inverse
Workers in the art of instrument design for instruments that could determine molecular weight worried that because the radius of gyration of the scatterer could not be easily measured and is not known in advance, the size factor P would not be known and could not be ignored without creating an error unless the scattering angle was very small which would make the size factor approximately 1 in equations (1) and (2). As a result, a design evolved in approximately 1970 for an instrument to measure molecular weight which was based upon low light scattering angles such that P could be ignored. Actually R.sub.9 can be measured, but it requires a measurement of R (Raleigh scattering) at each of a plurality of angles .theta.. M.sub.w (hereafter the weight averaged molecular mass M.sub.w may sometimes be symbolized as simply M.sub.w) and R.sub.9 can be derived from these measurements. Another instrument design in the prior art used this approach. For low angles .theta., R.sub.9 need not be known.
FIG. 1 is illustrative of this prior art design for a low angle light scattering detector (hereafter LAL) where R.sub.9 need not be known. A light source 10 which can be an arc source or a laser generates incident light which is focussed by optics 12 on the input window 14 of a flow cell 16. The flow cell is comprised of a long piece of input glass 18, a scattering volume 20 through which the scatterers eluting from a liquid chromatography column 22 flow in solution, a long piece of output glass 24, an output window 26, a mask 28 and a scattered light detector 30.
The LAL detector of FIG. 1 has many areas in which improvement can be made. First, the signal-to-noise ratio is not optimal in this design for several reasons. The light source 10, if an arc, generates light which is not well collimated and which must be focussed by the optics 12. As the light passes through the optics and enters the input window 14, the imperfections in the lenses and window cause some scattering of the incident light which, if it gets into the detector 30 represents noise since it is not light scattered by the scatterers but light scattered by the machine itself. Further, as the light exits the glass 18 and enters the scattering volume 20 and then re-enters glass 24 there is further scattering at the liquid-glass interfaces 32 and 34 caused by imperfections in the glass. Further scattering occurs at the output window 26 caused by imperfections in the glass there. All this scattered light is noise and not data and steps must be taken to eliminate it. One of these steps is to make the glasses 18 and 24 very long such that light scattered at the input window 14, and the interfaces 32 and 34 fairly far away from the detector 30 such that light scattered at these locations misses the detector. Light scattered at the output window 26 is somewhat masked from the detector 30 by the mask 28. FIG. 2 shows the prior art mask configuration for mask 28. The mask is comprised of an opaque center section 36 which blocks nonscattered incident light that passes straight through the flow cell and scattering volume without being scattered from entering the detector 30. The mask also has a concentric opaque outer section 38 which masks off high angle scattered light from entering the detector 30. The region 40 which is not cross-hatched is transparent and allows low angle scattered light to enter the detector regardless of whether it was scattered from the scatterers or by other things such as imperfections in the glass.
It is troublesome to use the structure of FIG. 1 because the flow cell must be disassembled and cleaned ultrasonically or otherwise, almost everyday to prevent dirt from collecting on the various surfaces in the optical path which could cause further scattering.
Further, the design of the optics 12 to focus the arc light is somewhat complicated and expensive if scattered light is to be minimized. With the advent of lasers, the problems of design of the optical system are lessened because lasers output collimated light. However, some optics must be present to "clean up" the laser beam to eliminate some scattered light. The problems of making the windows 14 and 26 and the interfaces 32 and 34 as perfect as possible are also quite difficult.
Another problem with the structure of FIG. 1 is that the output frequency of lasers that are relatively inexpensive (Helium-Neon lasers) is too far into the red end of the spectrum to make a good match for the band of highest sensitivity of the detector 30. Typically, the detector 30 is a photomultiplier tube (hereafter PMT) which is has a sensitivity which three times as efficient in the blue-green end of the spectrum as in the red end. PMT's optimized for the blue-green end of the spectrum also have lower dark current, i.e., unwanted signal when no scattered light is being detected, which results in less noise. Because the amount of scattered light compared to the incident light intensity is very, very small, noise considerations are extremely important in scattered light detector design and high intensity incident light is important to keep the intensity of scattered light at measurable levels. Further, the amount of light scattered is proportional to 1/.lambda..sup.4. Thus blue light which has a shorter wavelength .lambda. scatters much more than red light. A typical Helium-neon laser output is 633 nm. A typical line from an arc lamp which can be used is 467 nm. 633.sup.4 divided by 467.sup.4 is 3.4 which means the bluer light of he arc source scatters 3.4 times better than He-Ne laser light. Thus, it is highly desirable to use a PMT optimized for the blue-green. Lasers which output light in the blue-green end of the spectrum and have high output power are very expensive and very large. This makes the instrument bulky, heavy and expensive.
Further, lasers have ripple noise, i.e., noise modulated onto their output light intensity which is in the range from 0 to 30 Hz. Variations in the intensity of the incident light caused by the source translate into noise in the form of variations in the intensity of the scattered light not caused by the concentration or molecular weight of the scatterers. High performance liquid chromatography runs generally last from 30 to 90 minutes, and the output signals from the detectors are generally sample once per second. Thus, the sampling frequency is close to or within the frequency band of the ripple noise which tends to create further noise in the data.
Finally, another noise problem exists because of the inevitable presence of the very large particles in the output stream of the liquid chromatography column. These large particles possibly fines of column packing, and thus are not particles of interest, but, because of their large size, they cause a great deal of scattered light anyway. FIG. 3 illustrates the typical light scattering pattern of a small particle as a function of angle, and FIG. 4 illustrates the typical light scattering pattern of these large particles. In FIG. 3, the incident light vector I.sub.0 enters from the left and the scattered light vectors for light scattered at low angles and at approximately 90 degrees are represented by vectors I.sub.s0 and I.sub.s90, respectively. The intensity of light scattered at 90 degrees from small particles is approximately one-half the intensity of light scattered at low angles. Unfortunately, as seen in FIG. 4, the intensity of scattered light from large particles has a very different shape. FIG. 4 illustrates that very little light is scattered at 90 degrees from large particles, and most of the scattered light is forward at small angle. This means that these large particles, despite their relative scarcity, will cause large noise spikes in low angle scattered light detectors.
Accordingly, a need has arisen for a new design for a classical light detector and system for determining molecular weight of biological proteins and other small particles that enter a scattering volume of a flow cell.